Method for Making Lignocellulosic Paper and Paper Products

ABSTRACT

Enzyme compositions comprising laccase, lipase, cationic polymer, and optionally laccase activator, for papermaking application are disclosed. It also relates to the use of the enzyme composition to improve dry strength property of a paper product made from lignocellulosic-containing materials before or after mechanical refining in a papermaking process.

This application claims the benefit of U.S. Non-Provisional patentapplication Ser. No. 14/835,931, filed 26 Aug. 2015, the entire contentsof which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a method of making paper and paperproducts. More specifically, a laccase, lipase and cationic fixativepolymer composition is added to a lignocellulosic suspension to helpimprove dry strength of the paper and paper products.

Paper pulp is typically processed from wood through the Kraft processes.This process produces a cellulosic fiber with a dark brown color, mostlydue to the presence of lignin. For some applications, lignin moleculesare further removed by a process known as bleaching to produce bleachedfiber suitable for making paper products such as tissue, towel, andprinting and writing paper. For other uses such as linerboard,unbleached fiber is preferred because it is economical and alsoenvironmental friendly for not going through bleaching process usingtoxic bleaching chemicals. Unbleached Kraft fiber usually contains 1% to2% lignin. Although lignin is significantly reduced via the Kraftprocess, the remaining lignin is embedded in cellulose, resulting in alignocellulosic material that requires more than 50% of the energy thatis needed to refine a bleached fiber mechanically in papermakingprocesses. Other mechanical pulps such as thermal mechanical pulp (TMP)is another type of unbleached fiber that is widely used for papermaking.Lignocellulosic material is a term used to describe the wood fiber thatcontains lignin molecules. Many recycled brown furnishes are derivedfrom a mixture of different types of fibers with inferior quality thanvirgin fibers. Recycled fibers, e.g., old corrugated container (OCC) andwaste newspaper, not only contain lignin, hemicellulose and otherbiomass, but also contain a significant amount of contaminants known asstickies and pitches such as polyvinyl acetate and ester organiccontaminants. These types of contaminants can interfere with fiber tofiber bonding resulting in decreased dry strength of the final product.

To restore dry strength properties of the paper product made fromrecycled lignocellulosic material such as poor quality OCC fiber,papermakers traditionally use synthetic polymeric dry strengthadditives. The use of enzymes for papermaking has gained popularitysteadily due to the rapid developments of robust and inexpensive enzymeproducts and its environmentally friendly approach. Although cellulasesare being used recently for paper dry strength, the commercial successis limited to bleached virgin fiber or drinking pulp (DIP). It isevident that accessibility of cellulase to lignocellulosic fiber ishindered by lignin molecule and other non-cellulosic biomasses boundwith the cellulose. Although many commercial trials have been attempted,cellulase is generally not suitable for poor quality recycledlignocellulosic fiber, or short fiber TMP, etc., for dry strengthapplication. Until now, no enzyme technology has achieved significantcommercial success in papermaking with recycled OCC fiber, particularlypoor quality OCC. Thus, there is a need of an environmentally friendlyand sustainable enzyme approach for recycled OCC or unbleached virginfiber as an alternative technology or a replacement of the syntheticpolymeric additives.

Laccases are copper-containing enzymes that are known to be goodoxidizing agents in the presence of oxygen and are used for many otherapplications, including treatment of pulp waste water, pulp de-inking,industrial color removal, bleach for laundry detergents, oral care teethwhiteners. Laccases are being widely investigated for bio-bleaching woodfiber in pulping process as a replacement for toxic chemical bleachingreagents. Laccase is also capable of polymerizing lignin or polyphenolsin the wood fiber and thereby widely investigated as a catalyst or afacilitator to improve paper dry strength, either with or withoutmediators or radical generating chemicals. The likely mechanism for theimproved strength was the crosslinking of lignocellulosic fiber throughlignin oxidation and polymerization. In addition, laccase may alsooxidize other phenolic-containing components such as aromatic sidechains in protein, hemicellulose, cellulosic fiber, etc. under specificradical-assisted conditions, to provide functional groups that interactwith each other to give paper strength properties. Advantageously, theactions of laccase on lignin and other functional groups generally haveno adverse effect on fiber quality such as fiber length underconventional papermaking conditions.

U.S. Pat. No. 6,207,009 disclosed a process for producing paper orpaperboard from mechanical pulp in which the pulp is treated with aphenol-oxidizing enzyme, particularly laccase and peroxidase, aftermechanical refining of the pulp has been completed. The resulting paperexhibits an increased strength relative to paper produced from untreatedpulp. The prior art did not mention any synergistic effect of laccasewith lipase and cationic polymers for recycled lignocellulosic fiber.Similarly, U.S. Pat. No. 6,610,172 claimed a process for producing papermaterials having improved wet strength. This process involves (a)preparing a suspension of unbleached or semi-bleached chemical orsemichemical pulp or pulp from recycled fibers; (b) treating the pulpwith a phenol-oxidizing enzyme, e.g., laccase, and a mediator; and (c)de-watering the treated pulp in a papermaking machine to make paper.U.S. Pat. No. 5,603,804 described a process for producing linerboard orcorrugated medium using the oxidase-treated pulp. The pulp is unbleachedKraft pulp, neutral sulfite semichemical pulp, or recycled pulp from oldcorrugated containers or old news print. The oxidases include laccase,or catechol oxidase, or bilirubin oxidase.

US Patent Application No. 20140116635 described a method of making paperor paperboard having enhanced dry strength using a laccase or acellulase enzyme and a cationic water-soluble polymer. The prior art didnot disclose any synergistic effect of laccase with lipase and cationicpolymers on OCC recycled fiber.

Lipase or esterase has been commercially used for removing stickies orpitches adhered on the fiber surface in papermaking. Stickies contentvaries with fiber type and paper mill systems, and it poses a majorproblem to recycled paper mills, particularly to the Asian or Europeanlinerboard mills that routinely use poor quality recycled OCC. Not onlydo hydrophobic stickies or pitches accumulated on the process machineryto reduce productivity and/or deposit on paper products to lower paperproduct quality, but also do those hydrophobic organic contaminantsinterfere with cellulosic fiber-fiber interaction and thereby reducingpaper strength. In addition, those hydrophobic contaminants on fibersurface could prevent enzymes and chemical additives from accessing tofiber surface for reaction or interactions, and reduce the efficiency ofthese reagents.

US Patent Application No. 20070261806 disclosed methods of treating pulpstocks with an enzyme formulation containing one or more oxidativeenzymes, to reduce pitch deposition. It described that the pulp stock istreated with an enzyme formulation containing laccases, peroxidases,esterases, and/or combinations thereof. The enzyme formulations may alsocontain a laccase mediator and/or a dispersant. The enzyme formulationcan be applied at any of several locations during the pulping and/orpapermaking process, but typically applied as a solution to the pulpstock. The prior art did not discuss the effect of a cationic polymer onlaccase and esterase performances, and did not disclose any effect ofthe formations on paper dry strength property.

Cationic polymers could be used to blend with enzyme to improve enzymestability and accessibility of the enzyme to cellulosic fiber surfacevia their fixative property. It could also benefit in fiber retentionand COD reduction in recycled paper mills. Those benefits have beenproved in the lab and also in many commercial practices.

U.S. Pat. No. 8,454,798 disclosed a method for making paper or paperboard by applying a composition containing enzyme and cationic coagulantto papermaking pulp prior to paper forming. However, this prior art didnot disclose any synergistic effect of laccase, esterase and cationicpolymers on paper dry strength property.

US Patent Application 20140116653 disclosed a method of making paper orpaperboard having enhanced dry strength using an enzyme and a polymerincluding at least one of a cationic water-soluble polymer and anamphoteric water-soluble polymer. The prior did not disclose anyinformation on effect of esterase or lipase on paper strength.

It is known in the art that cationic polymer can be used in combinationwith enzyme for papermaking uses. Cationic polymers are used togetherwith enzymes for stickies control and strength applications. Thosecationic polymers includes poly(diallyldimethylammonium chloride),poly(DMA-Epi) polyamine, polyaminoamide derivatives and polyvinylaminederivatives etc. However, not all the cationic polymers would benefitenzymes performance or stability. As matter of a fact, many cationicpolymers reduce or deactivate activity of the enzymes such as laccaseand lipase. Polyvinylamine and glycoxylated PAM may covalently react andcrosslink enzyme to deactivate the enzyme activity completely. Simplycombining an enzyme with a cationic polymer is not a solution to all.

The current method provides a dry strength composition for papermakingto improve dry strength properties of a paper product and also improvethe efficiency of the papermaking process. It has been discovered that acombination of laccase and lipase together with cationic polymer with orwithout a laccase activator provides for synergistic effects inpapermaking and produces a paper product with improved dry strengthproperties. More specifically, the current method relates to the use ofa composition to improve dry strength properties of a paper product bytreating a pulp furnish containing lignocellulosic unbleached fiberand/or recycled brown stock.

In the current composition, laccase serves as an enzyme to polymerizelignin via oxidization, lipase catalyzes breakdown of organic stickiesand pitches on fiber surfaces and improves accessibility of laccase andthe fiber to fiber binding interaction. The cationic polymers help indispersing stickies, stabilizing the laccase and lipase and improvesfiber retention. When laccase and lipase were used in combination with acationic polymer, a synergetic effect was observed. Thus, the inventionprovides a three-component dry strength composition for use inpapermaking application.

BRIEF SUMMARY OF THE INVENTION

Provided is method of making a paper and paper products using a laccase,lipase, cationic fixative polymer composition as an additive to alignocellulosic suspension. More particularly, the current methodrelates to the use of a laccase, lipase and cationic fixative polymerformulation or composition to improve dry strength properties of a paperproduct made primarily of unbleached lignocellulosic fibers and/orrecycled brown stock. The dry strength composition includes, in additionto at least one cationic fixative polymer, at least one laccase having alaccase activity of at least 12 LAMU and at least one lipase having alipase activity of 0.1 to 10 KLU per kilogram (kg) of dry fiber. Theaddition of the dry strength composition to the lignocellulosicsuspension can be performed prior to, during, or after mechanicalrefining.

In the present application, it is believed laccase is an activeingredient that specifically catalyzes the oxidation of lignin,resulting in polymerization of lignin molecule. The laccase may alsocatalyze the oxidation of other phenolic components or carbohydratesunder specific conditions. The lipase of the composition can catalyzethe hydrolysis of the wood pitches such as fatty ester to enhanceaccessibility of laccase to fiber surface, or the hydrolysis and removalof stickies contaminants such as polyvinyl acetate from fiber to helpimprove fiber binding property. The three component dry strengthcomposition of the current method provides improved and enhancedperformances of paper dry strength relative to the use of one or two ofthe individual components alone. The present composition can also reduceorganic contaminants and improves turbidity of white water inpapermaking process. As used herein, enzyme composition is thecombination or mixture of one or more enzymes. By “dry strengthcomposition” it is meant the combination or mixture of laccase, lipaseand cationic fixative polymer.

Examples of laccases that can be used in the current method are NS51003and NS51002 from Novozymes (Bagsvaerd, Denmark); the optional laccaseactivator can be selected from copper sulfate, ascorbic acid andsalicylic acid; lipases such as StickAway® or Resinase® A2X fromNovozymes (Bagsvaerd, Denmark), and cationic fixative polymer such asthose available from Solenis LLC (Wilmington, Del., USA) including ZenixDC® 7429 and Zenix® DC7479.

It should be noted that copper ion may be important for laccase'scatalytic activity or enzyme stability. Laccase from a commercialsources could lose its activity when the copper ion is stripped awayfrom the tertiary structure of a laccase protein. It was discovered thatlaccase can lose its activity quickly upon dilution with water,especially at elevated temperatures. This may be explained by thepossibility that the copper ion is released from laccase when the enzymesolution is diluted. It was also discovered that the addition of a smallamount of copper sulfate to a laccase formulation helped maintain thelaccase activity upon dilution. With additional copper ion in theformulation, the equilibrium of copper ion shifts to the laccase proteinso the tertiary structure of the enzyme is maintained in a stable form.Other ingredients of the dry strength composition may also extract thecopper ion away from laccase so additional copper sulfate may be neededfor the enzyme composition to maintain laccase activity. It was foundthat activity of laccase improved significantly when the copper sulfatewas added to the enzyme composition in the range of 0.05 to 0.1 wt. %.However, when the copper level was further increased to 0.5 wt. %, thelaccase lost some of its original activity.

The dry strength composition used in the current method is an aqueousformulation, typically containing up to 95% of water and 5-50% of othernon-aqueous components.

In one embodiment of the dry strength composition, the active content ofwherein the laccase content is from about 3 wt. % to about 40 wt. % andcan be about 10 wt. % to about 25 wt. % by total weight of thecomposition; the lipase content is from about 1 wt. % to about 80 wt. %,can be from about 3 wt. % to about 40 wt. % and may be from about 5 wt.% to about 20 wt. % by total weight of the composition; and the cationicfixative polymer content can be from about 2 wt. % to 50 wt. %, can befrom about 5 wt. % to about 40 wt. %, and may be from about 7% to about20% by total weight of the composition.

Laccase alone or in combination with a cationic fixative polymer, may beused in papermaking processes to improve paper properties. However, notall the cationic fixative polymers are compatible with laccase. It wasfound through laccase assays that some cationic fixative polymers canreduce or even deactivate laccase NS51003 (a laccase from Novozymes).Those cationic polymers include polyvinylamine and glyoxalatedpoly(acrylamide) that may have covalently reacted and cross-linked withlaccase to deactivate the enzyme activity. It was also discovered thatthe enzyme composition of the current method performed better than acombination of laccase and cationic polymer in providing improved drystrength to a paper product made from recycled OCC, particularly fromthe poor quality OCC that contains lots of stickies and pitchescontaminants.

The current method also relates to the process of making a paper productusing a dry strength composition of laccase, lipase and a cationicpolymer. In some aspects, a lignocellulosic fiber in an aqueous solutionis formed to produce a pulp slurry. The dry strength composition isadded to the pulp slurry and the slurry is dewatered and dried toproduce the desired paper product. The lignocellulosic fiber in anaqueous solution as used herein is described as a pulp slurry, pulpfurnish or pulp suspension, all of which mean the same thing.

The dry strength composition of the current method can be formulated atdifferent weight ratios of laccase and lipase depending on the specificpulp furnish. In general, an enzyme composition with higher weight ratioof laccase to lipase gave better strength results for an unbleachedvirgin fiber or a good quality old corrugated container (OCC) furnishhaving Canadian standard freeness (CSF) higher than 500, while theenzyme composition with higher ratio of lipase vs. laccase gave betterstrength results for a poor quality recycled OCC furnish with freenessless than 400 CSF.

The dry strength composition of the current method may be also used toreduce organic contaminants in papermaking process and improvepapermaking productivity. The cationic fixing polymer is effective ininteracting with anionic trash, dispersing stickies and pitch particles,and helping improve fiber-to-fiber interaction and flocculation whichcould result in better drainage. It was found that the treatment of therecycled fiber with the enzyme composition had no negative effect onfiber yield of virgin unbleached fiber, and the enzyme compositionimproved fiber retention and white water turbidity of a recycled OCCfurnish.

The enzyme compositions have shown synergistic effect in improvinglaccase activity and papermaking performance for enhanced dry strengthproperties of paper product made from lignocellulosic material,particularly recycled OCC fibers.

DETAILED DESCRIPTION OF THE INVENTION

The current method relates to paper products having improved drystrength. More particularly, the method relates to a composition formaking a paper product that comprises laccase, lipase, cationic fixativepolymer and optionally laccase activity modifiers or activators whereinthe laccase content is from about wherein the laccase content is fromabout 3 wt. % to about 40 wt. % and can be about 10 wt. % to about 25wt. % by total weight of the composition; the lipase content is fromabout 1 wt. % to about 80 wt. %, can be from about 3 wt. % to about 40wt. % and may be from about 5 wt. % to about 20 wt. % by total weight ofthe composition; and the cationic fixative polymer content can be fromabout 2 wt. % to 50 wt. %, can be from about 5 wt. % to about 40 wt. %,and may be from about 7% to about 20% by total weight of thecomposition.

In other aspects, the current method relates to the use of enzymes toimprove dry strength properties of a paper product. The method relatesto the addition of a composition to a pulp furnish or suspension, suchas a pulp furnish containing unbleached fibers or recycled brown stock,wherein the composition comprises a laccase with an activity of at least12 LAMU and a lipase having a lipase activity of 0.1 KLU per Kg to 10KLU per Kg of dry fiber, and wherein the enzymes are added to thepapermaking process either before, during or after mechanical refiningin a papermaking.

The laccase of the current method may be derived from microbial, fungal,or other sources. It may furthermore be produced by recombinanttechniques. The laccase of the current method can be from a commercialsource, for example, NS51003 and NS51002 from Novozymes (Bagsvaerd,Denmark).

The laccase of the current method can also include enzymes that possesslaccase activity based on current assay methods. The activity of thelaccase used in the Examples below were determined using syringaldazineas the substrate or by the ABTS assay.

Examples of enzymes containing laccase activity include, for example,laccase (EC 1.10.3.2), catechol oxidase (EC 1.10.3.1), mono-phenolmonooxygenase (EC 1.14.99.1), bilirubin oxidase (EC 1.3.3.5), andascorbate oxidase (EC 1.10.3.3). These can be used alone or incombination with one another. The EC (Enzyme Commission) number is basedupon the Nomenclature Committee of the International Union ofBiochemistry and Molecular Biology (IUBMB).

In other aspects of the current method, the laccase modifier oractivator can be one or more inorganic or organic compounds, such ascopper sulfate, copper ion salts, other metal ions salts, and ligandsthat help activate laccase activity, and also laccase mediators oractivators including ascorbic acid, ascorbate, salicylic acid,salicylate, nicotinic acid, nicotinate, a hardwood black liquor, asoftwood black liquor, ligno-organosolv, lignin sulfonate, 2-thiouracil,N-benzylidene-benzylamine, melamine, ferric chloride, potassiumferricyanide, guanidine, cyanuric acid, nicotinic acid, pyruvic acid,imidazole, phenol, and mixtures thereof. The term laccase modifier,laccase mediator, laccase activator, and laccase enhancer are usedinterchangeably and relate to the same compounds.

In yet another aspect of the current method, the laccase enhancer can becopper sulfate, ascorbic acid, salicyclic acid and combinations thereof,at a dosage of from about 0.01 wt. % to about 0.5 wt. % by weight of thetotal composition. The activity may be negatively affected by a highlevel of copper sulfate at >0.5% based on the total weight of the drystrength composition.

It should be noted that laccase needs oxygen to be active. Therefore,effective oxygen and air flow in the papermaking process helps toimprove the enzyme activity and efficiency of the laccase in thepapermaking application.

In some aspects of the current method, lipases can be derived frommicrobial, fungal, or other natural sources. Lipases can also beproduced by a genetic recombinant technique or via chemicalmodifications. The enzymes possessing lipase activity include, forexample, tri-alkanoate glycerol lipase, fatty ester lipase, esterase,phospholipase, or combination thereof. Commercially available enzymescontaining lipase activity include, for example, Stick Away® orResinase® A2X, Resinase® NT, Palatase® from Novozymes (Bagsvaerd,Denmark), and Lipase G-1000 from DuPont Industrial Biosciences (PaloAlto, Calif., USA). The lipase activity in the following examples weredetermined using the standard lipase KLU (KLU equals to 1000 lipaseUnits, defined in WO 89/04361) or determined by lipase assays describedin the current method.

The lipases of the current method also include enzymes that possesscatalytic activity of hydrolyzing ester bonds, based on the assays ofthe current method. Enzymes, such as proteases and amidases are known tocontain lipase activity and therefore could be used in the currentmethod.

In one aspect of the present method, current method the lipase has highesterase activity as determined by lipase assay using triacetin as asubstrate. This lipase preferably catalyzes the hydrolysis ofhydrophobic polyvinyl acetate to release hydrophilic polyvinyl alcoholand acetic acid. One lipase is StickAway®, which possesses strongesterase activity towards triacetin but also lipase activity towards afatty ester with a long chain alkyl group up to C18 carbons.

When StickAway® was added to a polyvinyl acetate contaminated oldcorrugated container (OCC) suspension, the paper product made from thetreated fiber achieved more than 10% improvement in strength propertiesover the control without lipase treatment (see Table 1).

It is envisioned any enzymes containing lipase activity towards shortchain alkyl esters can also work to enhance fiber binding property andpaper strength. For example, it was discovered that Resinase® A2X workedas well as StickAway® with North American OCC furnish (see Table 2).

In other aspects, the cationic fixative polymers used in the currentmethod with the laccases and lipases can be selected from the groupconsisting of poly(diallyldimethylammonium chloride),poly(dimethylamine-epichlorohydrin-ethylene diamine), cationicpoly(acrylamide), poly(ethyleneimine), hydrophobically modified cationicpolymers, long chain alkyl glycidyl ether modified poly(aminoamide),cationic natural products such as, cationic starch and cationic guar,amphoteric polymers that are net cationic, and combinations thereof.Other cationic fixative polymers that can be used in the current methodare commercially available from Solenis LLC, Wilmington, Del., USA, suchas Zenix DC® 7429, Zenix® DC7479 and DeTac® DC786C. The cationicfixative polymers of the current method can be one or more papermakingadditives such as a dry strength resins, wet strength resins,flocculants, retention aids, and/or drainage aids. It is worth notingthat a different cationic polymer can be applied to a papermaking systemin combination with the current dry strength composition to improveoverall performance of the papermaking process. It should also be notedthat not all the cationic fixative polymers are suitable for laccase andsome cationic polymers, such as polyvinylamines and glycoxylatedpolyacrylamides can reduce or even deactivate the activity of laccase.For example, a polyvinylamine based cationic polymer negatively affectsactivities of laccase or lipase when the polymer is blended with theenzymes, but as long as the polymer is not blended directly with theenzymes, it could be used in combination with the laccase and lipaseenzymes in the papermaking process.

In some aspects of the current method, the dry strength composition canbe stabilized by one or more compounds selected from propylene glycol,glycerol, ethylene glycol, sorbitol, lactic acid, glucose, galactose,maltodextrin, monosaccharides, oligosaccharides, corn syrup, inorganicsalts such as sodium and potassium chloride, a pH buffer system such assodium or potassium phosphates, sodium citric acid,tris(hydroxymethyl)methylamine (Tris),4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES);piperazine-N,N-bis(2-ethanesulfonic acid), and 2 2-(N-morpholino)ethanesulfonic acid.

In yet another aspect of the method, the dry strength composition of thecurrent method includes at least one laccase, at least one lipase withhigh esterase activity as determined by the lipase assay using triacetinas substrate, at least one cationic fixative polymer selected frompoly(diallyldimethylammonium chloride),poly(dimethylamine-epichlorohydrin-ethylene diamine), and mixturesthereof, and optionally a laccase activator, such as copper sulfate,ascorbic acid, salicyclic acid and combinations thereof.

The weight ratio of laccase/lipase/cationic polymer of the dry strengthcomposition of the current method is important for its performance inpapermaking as a strength additive. The ratio of these three mainingredients of the dry strength composition of the current method can bechanged to a specific range to provide optimized enzyme activities andstability under specific pH, ionic strength and temperature conditions.The percentage levels of the three ingredients also affect laccase andlipase efficiencies of treating different types of unbleachedlignocellulosic fibers to improve paper dry strength. The dry strengthcomposition of the current method is an aqueous formulation, typicallycontaining up to 95% of water and 5-50% of other non-aqueous components.In one embodiment of the dry composition, the active content of alaccase is from wherein the laccase content is from about 3 wt. % toabout 40 wt. % and can be about 10 wt. % to about 25 wt. % by totalweight of the composition; the lipase content is from about 1 wt. % toabout 80 wt. %, can be from about 3 wt. % to about 40 wt. % and may befrom about 5 wt. % to about 20 wt. % by total weight of the composition;and the cationic fixative polymer content can be from about 2 wt. % to50 wt. %, can be from about 5 wt. % to about 40 wt. %, and may be fromabout 7% to about 20% by total weight of the composition.

The active weight percentage of the laccase and lipase of the drystrength composition is defined on the basis that the commercial enzymesare 100% active as they are obtained from a commercial source. Theactive percentages of the cationic fixative polymer and laccaseactivator of the composition are defined as non-aqueous parts of thesepolymers or chemicals of the dry strength compositions.

The enzyme composition of the current method exhibited improved laccaseactivity relative to the original laccase. The term “improved laccasestability” is intended to indicate that the enzyme composition afterbeing stored for a period of time at a certain temperature, and issubjected to the same standard test conditions as the original laccaseat the same dilution factor, exhibits less in reduction of the laccaseactivity compared with that of the original laccase.

In the enzyme composition of the current method, lipase activity wasmeasured using triacetin or tributyrin as substrates via the titrationmethod as described in the example section. It was found that thecationic fixative polymer Perform® PC8229 and/or the laccase NS51003 hadno negative effect on the lipase activity of StickAway®. The lipaseactivity of the composition was relatively stable.

In some aspects of the current method, the pH of the dry strengthcomposition can be from about 3 to about 10, can be from about 4 toabout 9, and can be from about 5 to about 8. In still other aspects ofthe method, laccase can be optionally mixed with a laccase activator for5 to 30 minutes at room temperature followed by the addition of thelipase and cationic fixative polymer. However, in other aspects, theingredients can be added in any sequence in the process of formulatingthe composition prior to the composition being added to the pulpfurnish. The pH adjustment of the formulation can be done at the end ofthe process with an acid or an alkali after all the ingredients become ahomogenous formulation. A buffer system may also be used to control thepH of the enzyme composition within a specific range.

Physical storage stability is a factor when evaluating the properties ofthe dry strength composition of the current method. The term “goodphysical stability” of the product is intended to indicate that theenzyme composition has maintained desired physical properties inappearance, homogeneity and having no deteriorated odor. The weightratio of the cationic fixative polymer is one of the factors affectingsuch a stability.

The laccase enzyme activity of the laccase used in the current method,was measured by a standard syringaldazine assay as described in theexperimental section. The activity was in the range from about 200Laccase Myceliophthora Units (LAMU) to 10,000 LAMU per gram, can be fromabout 500 to about 5,000 LAMU per gram, and may be from about 1,000 to2,000 LAMU per gram. The lipase activity of the enzyme used in thecurrent method, is defined in WO 89/04361, and was in the range of fromabout 2 KLU/gram to 50 KLU per gram (one KLU is equal to 1,000 lipaseUnits), can be from about 5 KLU to about 25 KLU per gram, and may befrom about 10 KLU per gram to about 30 KLU per gram. The laccase andlipase activities of the enzymes used in the dry strength compositionmay vary with specific batches of products and the commercial sourcesfrom where the enzyme came from. However, amounts used in theexperiments were calculated based on the assumption of being 100% activeas received.

In other aspects of the current method, the laccase activity of the drystrength composition of the current method is normally in the range offrom about 40 LAMU per gram to about 2,000 LAMU per gram, can be fromabout 100 LAMU per gram to about 1,000 LAMU per gram, and may be fromabout 200 LAMU per gram to about 400 LAMU per gram. The lipase activityof the lipase used in the current method is normally in the range offrom about 0.1 KLU per gram to about 15 KLU per gram, can be from about0.25 KLU per gram to about 10 KLU per gram, and may be from about 0.5KLU per gram to about 5 KLU per gram. The enzyme activities of the drystrength composition may be evaluated under specific pH and temperatureconditions with different enzyme substrates as needed.

In some aspects of the current method, the dry strength composition maybe used in treating all types of cellulosic fibers, such aslignocellulosic fiber including bleached, unbleached virgin fiber,mechanical fiber and OCC recycled fiber. In some aspects of the currentmethod, the dry strength composition may be used to treat a mixture ofbleached fiber, unbleached virgin fiber and recycled fiber at a certainfiber mixing ratio. In other aspects, the dry strength composition ofthe current method is useful in providing improved dry strengthproperties of recycled linerboard produced in papermaking. The drystrength composition may work effectively with poor quality recycledfiber from Asian such as TOCC (Taiwan OCC), COCC (Chinese OCC), EOCC(European OCC), and better quality AOCC (American OCC) as well asunbleached Kraft fiber (UBSK). The degree of the improvement in aspecific strength property varies with fiber type and treatmentconditions and the specific enzyme composition.

It was found that the dry strength composition of the current methodusually provided higher improvement in Ring Crush to the paper made froma good quality fiber such as AOCC and UBSK while better performance wasobserved in dry tensile and Mullen Burst properties of the paper madefrom the poorer quality fibers such as TOCC or COCC or EOCC. Inaddition, the enzyme composition having a higher weight ratio of laccasevs. lipase is more efficient in improving ring crush and other strengthproperties than a better quality AOCC and unbleached virgin fiber. Thecomposition having a higher weight ratio of lipase vs. laccase gavebetter results in treating poor quality OCC from Asian and Europe thatcontained high levels of stickies and pitches.

In another aspect of the current method, lignocellulosic fiber insuspension is treated for at least 0.1 hours with the dry strengthcomposition wherein the dry strength composition has at least 12 LAMU oflaccase activity and 0.1 to 10 KLU of lipase activity per Kg of dryfiber and the fiber suspension is at a temperature of from about 20° C.to about 70° C. and a pH of from about 4.0 to about 9.0. The treatedfiber suspension can optionally be refined using a mechanical refinerfor wood fiber either prior to or subsequent to the addition of the drystrength composition. The treated suspension can then be dewatered anddried to form the desired paper product. The dry strength properties ofthe paper product, such as Mullen burst, dry tensile, Ring Crush, ZDT,etc. are tested and the data normalized based on the basis weight of theblank sheet with treatment or the control with the individualingredients of the enzyme composition.

In yet another aspect of the current method, the pH of the treated pulpfurnish is from about 3.0 to about 9.0, can be from about 4.0 to about8.5 and may be from about 4.5 to 8.0; contact time of the dry strengthcomposition with pulp furnish is from about 0.1 hour to about 5 hours,can be from about 0.2 hours to about 3 hours and may be from about 0.3to about 2 hours; The temperature can be in the range of from about 10°C. to 70° C., can be in the range of from about As the stocktemperature, pH and other conditions in a papermaking system varies withpaper machines and specific fibers, the efficiencies of laccase andlipase in a specific enzyme formulation may vary as well as theirparticular performances.

The enzyme composition of the current method can introduced into apulper during the pulping stage, or brought into contact at any stockstorage chest, high consistency chest or other holding tank. It can alsobe added into the paper machine white water or, alternatively, can beapplied in the water treatment loops of virgin or recycling mills totreat wood fiber. An effective agitation or mixing is needed for thelaccase and lipase to have an effective action on the fiber. Air flow inthe papermaking system is particularly critical for laccase that needsoxygen to be active. Adding an oxidizing agent, such as oxygen, orhydrogen peroxide, and other peroxides, or TEMPO reagent, may helpimprove laccase efficiency in the oxidation reactions. The pulpconsistency is also a factor for the effectiveness of the treatment bythe enzyme composition. High pulp consistency reduces mass-transferefficiency, resulting in non-uniform interactions between the enzymecomposition and fiber. Low pulp consistency decreases the concentrationof the enzymes in the pulp at the same dosage of the enzyme compositionbased on dry fiber and reduces enzyme efficiency. In general, the pulpconsistency of the lignocellulosic fiber treated by the enzymecomposition of the current method is in the range of 0.3% to 5%,preferably in the range of 0.5% to 4%, and most preferably in the rangeof 1% to 3%.

In some aspects of the current method, the laccase, lipase, the cationicfixative polymer, and optionally laccase activator of the dry strengthcomposition can be formulated together providing a stable composition.In other aspects, the three or four ingredients can be used in anycombination, added to the pulp furnish separately, be added at the sameor different points in the papermaking process, and can be added in anysequence to the pulp furnish to realize the strength benefits of thecomposition.

The improved laccase activity was observed with the combination oflaccase, laccase activator, lipase and cationic fixative polymer viaABTS laccase assay. The results have revealed that the cationic fixativepolymer improved the laccase activity and lipase gave furtherimprovement on the laccase activity than seen with the addition of thecationic polymer alone.

The dry strength composition of the current method may be used incombination with other papermaking performance additives to improvepaper product properties, such as cationic, anionic, amphoteric, anonionic synthetic compounds and natural polymers. Examples of compoundssuitable for use with the composition of the current method include, butare not limited to, dry strength papermaking additives, such as starch,starch derivatives, polyacrylamide derivatives, guar, poly(vinylamine),contaminant control detackifiers or fixative detackifiers, such asnonionic or anionic detackifiers, hydrophobically end-cappedpoly(ethylene glycol), poly(vinyl alcohol-vinyl acetate), whey protein,soy protein, hydrophobic and hydrophilic block copolymers,hydrophobically modified hydroxyethyl cellulose, wet strengthpapermaking additives including, but not limited to polyethyleneimine,urea formaldehyde resin, epichlorohydrin reacted poly(aminoamide),starch aldehyde, glyoxalated poly(acrylamide); flocculants for watertreatment; coagulants for water treatment; drainage aids forpapermaking; retention aids for papermaking; sizing agent for paperproducts; adhesives; debonders; softeners; creping adhesives;plasticizers for optimizing resin properties; and modifiers foroptimizing resin properties. Individual components of any of the abovecombinations may be applied together or sequentially in papermaking.Additionally, individual components listed above may be used incombination or blended together prior to use to make stable formulationsor they can be combined on site at a paper mill prior to use.

In some aspects of the current method, the dry strength composition maybe used in combination with one or more other enzymes such ashydrolases, cellulases, xylanases, proteases, amylases, hemicellulases,mannanases, pectinases, lyases, such as pectate lyase, cutinase,oxidoreductases, such as glucose oxidase and peroxidases, or anycombinations thereof. These enzymes can be used in any form, such as inliquid or solid form. Individual enzymes or any combinations ofdifferent enzymes may be applied together with the dry strengthcomposition of the current method, or applied sequentially before orafter the addition of the dry strength composition of the currentmethod. Individual enzymes may be also blended together with the drystrength composition of the current method to form a blended compositionprior to use.

An experimental model was established to simulate a real situation inrecycled paper mills. Polyvinyl acetate, as model stickies, may becoated onto OCC paper at 1-2 weight % (based on dry wt. fiber) and thecoated fiber may be pulped to make a uniform furnish for the drystrength treatment and the subsequent papermaking. It was seen that theOCC fiber pulped from the coated paper had higher paper dry strengththan the blank after the OCC fiber was treated with the enzymecomposition of the current method or a lipase.

EXAMPLES

The following examples further illustrate the current method, and theyare not intended to be in any way limiting to the scope of the method asclaimed.

Determination of Laccase Activity

Laccase activity was determined using syringaldazine as a substrate. Inthis assay, a laccase containing sample was incubated withsyringaldazine dissolved in methanol under aerobic conditions in 0.1molar (M) phosphate buffer at pH 7.5 and 25° C. for 110 seconds. Thesyringaldazine was oxidized to tetramethoxyl azo bis-methylene quinonehaving a molar absorptivity of 65,000 at A540 nanometer (nm). Theabsorbance was measured at 540 nm for 50 seconds. The standard laccaseenzyme unit (LAMU) is the amount of enzyme which converts lmicromole(μmol) syringaldazine to its quinone form per minute under theprescribed reaction conditions. One of the laccases used in the examplesbelow is NS51003 from Novozymes (Bagsvaerd, Denmark) that has a laccaseactivity unit no less than 1,000 LAMU per gram as reported and measuredat 1,050 LAMU per gram. The laccase activity can vary with batches,storage time and storage temperature.

ABTS Laccase Assay for Relative Laccase Activity

The laccase activity was also determined using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) as asubstrate. One unit of activity is equal to the micromole of theoxidized product from ABTS per min per mg protein at pH 4.0 to 6.0 at23° C. in an acetate buffer. The extinction coefficient of the oxidizedABTS had a molar absorptivity of 30,000 at A420 nm. A diluted enzymesolution (1.5 milliliter (ml)) was added to a mixture of 1.5 ml ABTS(0.5 millimole (mM)) solution and 1.5 ml of sodium acetate buffer (1 mM)to initiate the oxidization reaction. After mixing, incubation wasconducted at 23° C., while the change in absorbance per minute wasmeasured at 420 nm. Two Aspergillus laccases were used both fromNovozymes (Bagsvaerd, Denmark), to compare with the enzyme compositions.The assay pH was found to have had an effect on laccase activity.NS51002 alone worked best at pH 4-5 while NS51003 worked best at pH 5-6.

Lipase Assay to Differentiate Esterase and Lipase for Short Chain andLong Chain Alkanoate Esters

Lipase activity was determined using tri-alkanoate glycerol as asubstrate. One unit of activity is equal to a micromole of alkanoic acidreleased in 1 minute by 1 gram enzyme at pH 7.0. A one gram sample oftri-alkanoate glycerol was added to 50 grams of a 0.2 molar (M) sodiumchloride solution containing 10 microliter (μ1) of 1% phenolphthalein inethanol at 45° C. A lipase solution (0.01 g) was added to initiate ahydrolytic reaction. While stirring, the pH was maintained at 7.0 byadding 0.1M NaOH solution to give a slightly pink color (or using apH-Stat). The total amount of NaOH solution consumed in 5 to 10 minuteswas used to calculate the alkanoic acid released in the reaction perminute. In this type assay, triolein was used as a substrate for thelipase with activity to hydrolyze long chain alkanoate ester andtriacetin was used as a substrate to measure esterase activity tohydrolyze short chain alkanoate ester. Tributyrin was also used as asubstrate to measure both lipase and esterase activities. For example,as measured using triolein and triacetin as the substrates, StickAway®had 2.5 times more esterase activity than Resinase® A2X when triacetinwas used as the substrate while StickAway® was only 44% of lipaseactivity of Resinase® A2X when triolein was used as the substrate.

The enzyme activity was also determined using p-nitrophenol esters ofethanoate, butanoate, dodecanoate, and hexadecanoate as substrates. Oneunit of activity is equal to μmol of p-nitrophenol released in 1 minuteby 1 gram enzyme solution at pH 7.5 and 30° C. Substrate solutions (50mM) of p-nitrophenol esters were dissolved in dimethyl sulfoxide priorto addition to the reaction mixture. The assay was initiated when thesubstrate solution was added to 50 mM sodium phosphate buffer (pH 7.5)solution containing lipase activity. Initial rates of p-nitrophenolrelease from the substrates were quantitated by measuring absorbance at410 nm with molar absorptivity of 12.2 at pH 7.5.

One of the lipases used in the current method is StickAway® fromNovozymes (Bagsvaerd, Denmark) that has the standard lipase at 16.4KLU/g (KLU equals to 1,000 lipase Units, as defined in WO 89/04361). Theactivity can vary with batches, storage time and temperature.

Protein Assay

The protein concentration was determined using the Bio-Rad Protein AssayMethod, which is a dye-binding assay based on the method of Bradford andinvolves the addition of an acidic dye to a protein solution, andsubsequent measurement at 595 nm with a Jenway 6320D spectrometer.Comparison to the bovine serum albumin (BSA) standard curve provides arelative measurement of protein concentration. The Bio-Rad protein assayreagent was obtained from Bio-Rad Laboratories. The protein standard wasbovine serum albumin (BSA).

The protein assay was used to measure protein content in percentage ofthe dry strength composition of the current method and to determine thespecific enzyme activity.

Example 1—Synergetic Effect of Laccase, Lipase and Cationic PolymerCombination on OCC Paper Strength

Example 1, demonstrates improvement in Mullen burst and Ring Crush paperdry strength properties of paper sheets made from 100% recycled OCC. TheOCC medium fiber was pulped in water to 3% consistency creating a pulpslurry and refined to 320 milliliter CSF using a valley beater. Theresulting pulp slurry was treated with laccase NS51003, StickAway® andcationic fixative polymer Perform® PC8229, each being added to theslurry individually and also in combinations at 50° C. for 60 minutesunder effectively stirring. The dosages of the chemicals used for thetreatment were based on the dry fiber in percentage. The combination ofchemicals were mixed together prior to the addition to the pulp slurry.After the treatment, the pulp slurry was cooled down to room temperatureusing an ice water bath. Paper handsheets having a basis weight of 80lb./3000 sq. ft. were made on a Noble and Wood hand sheet machine at pH7.0. Mullen Burst (TAPPI Test Method T403) and Ring Crush (TAPPI TestMethod T818) were determined, and expressed as % versus the control inTable I.

TABLE I Synergetic effect of NS51003, StickAway ® and Perform ® PC 8229on OCC paper strength. Laccase Stick- Perform ® Ring Mullen N551003Away ® PC8229 Crush Burst Examples Dose % Dose % Dose % % % Comparative0.2 0 0 105.1 103.6 example 1-1 Comparative 0 0.2 0 101.0 105.9 example1-2 Comparative 0 0 0.2 92.5 102.2 example 1-3 Comparative 0.2 0.2 0104.9 107.5 example 1-4 Comparative 0.2 0 0.2 104.9 109.1 example 1-5Comparative 0 0.2 0.2 98.2 112.5 example 1-6 Example 1-1 0.2 0.2 0.2108.6 121.5 Example 1-2 0.1 0.1 0.1 105.4 117.5

In Table I, NS51003 from Novozymes was the laccase, StickAway® was thelipase also from Novozymes and Perform® PC8229, apoly(diallyldimethylammonium chloride) from Solenis LLC was the cationicfixative polymer. The results indicate improved dry strengthperformances for the combination of laccase NS51003, lipase StickAway®and cationic fixative polymer Perform® PC8229 (Example 1-1 and 1-2) inboth Mullen burst and Ring Crush compared with those individualcomponents alone and in all the other combinations when only two of thethree chemicals were used (Comparative Example 1-1 to 1-6). The drystrength composition containing the laccase, lipase and polymer (Example1-1) gave 21.5% improvement in Mullen Burst over the blank at the sameenzyme and polymer dosages and 17.5% improvement at 50% reduced enzymesand polymer dosages (Example 1-2). This clearly demonstrates thesynergistic effect of the combination of the three components on Mullenburst. Dry strength improvement in the Ring Crush test with the threecomponent system (Example 1-1) provided an 8.6% increase when comparedwith the laccase, lipase and polymer being added independently or incombinations of only two of the chemicals using the same enzyme andpolymer dosages.

Example 2—Formulation Process of the Enzyme Compositions

Example 2, illustrates a method of preparing the dry strengthcomposition of the current method using laccase, lipase, cationicfixative polymer and a laccase activator.

A laccase, optionally a laccase activator when needed, and a lipase wereadded sequentially to water at about 20° C. with gentle stirring untilbecoming a homogenous solution. A solution of the cationic fixativepolymer was added to the homogenous solution over 20 minutes at roomtemperature. The temperature of the resulting solution was maintained atabout 20° C. and stirred for 20 minutes and then the pH was adjusted to7.0 using HCl or NaOH. The solution was a homogenous brown color. Theactive content in weight percentage of the laccase or lipase (alsotermed as laccase active′ or ‘lipase active’) of the enzyme compositionwas based on the original enzyme at 100% active as it is obtained from acommercial source. The active content in weight percentage of thelaccase activator or cationic fixative polymer (also termed as ‘polymeractive’) of the dry strength composition is defined as the non-aqueousparts of the components of the dry strength composition. The Bio-Radprotein assay was used on the dry strength compositions to determineprotein concentration of the enzyme composition. Some of therepresentative enzyme compositions are tabulated in Table II.

TABLE II Formulation of the dry strength compositions of laccase, lipaseand cationic polymers Cationic Appearance fixative Laccase Appearanceafter 30 days Examples Laccase Lipase polymer activator as made At32-35° C. Example NS51003, StickAway ® Perform ® None 1-1 33% 33%PC8229, 33% Example NS51003, StickAway ® Perform ® None Homogenous Nochange 2-1 24% 6% PC8229, 20% Example NS51003, StickAway ® Perform ®CuSO4, Homogenous No change 2-2 24% 6% PC8229, 20% 0.05% ExampleNS51003, StickAway ® Perform ® None Homogenous No change 2-3 18% 12%PC8229, 20% Example NS51003, StickAway ® Perform ® None Homogenous Alittle 2-4 15% 15% PC8229, 20% settlement Example NS51003, Resinase ®Perform ® None Homogenous No change 2-5 15% 15% PC8229, 20% ExampleNS51003, StickAway ® Perform ® CuSO4, Homogenous A little 2-6 18% 12%PC8229, 20% 0.05% settlements Example NS51003, StickAway ® Perform ®None Homogenous No change 2-7 18% 12% PC8229, 10% Example NS51003,Resinase ® Perform ® None Homogenous Settlement 2-8 18% 12% PC8229, 40%Example NS51003, StickAway ® Zenix ® None Homogenous No change 2-9 15%15% DC7479, 20% Example NS51003, Resinase ® Perform ® AscorbicHomogenous 2-10 18% 12% PC8229, 20% acid 0.5% Example NS51003,StickAway ® Perform ® Salicylic Homogenous 2-11 18% 12% PC8229, 20% acid0.5% Example NS51003, StickAway ® Perform ® Homogenous 2-12 40% 40%PC8229, 20%

The compositions in Table II also contain 20% glycerol and water, unlessotherwise noted, to make up 100% in total weight. Example 1-1 andExample 2-12 do not contain glycerol. Zenix® DC7479 ispoly(dimethylamine-epichlorohydrin-ethylene diamine), a cationicfixative polymer from Solenis LLC.

Example 3. Synergy of the Enzyme Composition for Improved LaccaseActivity

The ABTS laccase assay was used to evaluate the effects of cationicfixative polymer, lipase and the laccase activator of the dry strengthcompositions on laccase activity. The same amount of laccase active ineach composition was used in the assay. In Table III, the relativeactivity numbers were determined by normalizing the values based on thatof laccase alone at 100%. The effects of lipase StickAway® and cationicpolymer Perform® PC8229 on the ABTS colorimetric assay were small butwere also measured and included in the calculation.

TABLE III Effect of additives on Laccase activity based on Laccase assayLaccase Activity Enzyme Compositions (ABTS Examples Description Assay) %Comparative Laccase NS51003 only 100 Example 3-1 Comparative LaccaseNS51003 104.5 Example 3-2 and Perform ® PC8229 18:20 blend Example 2-3Enzyme composition 108.2 Example 2-10 Enzyme composition with 115.5ascorbic acid Example 2-11 Enzyme composition with 115.2 salicylic acidExample 3-1 Example 2-3 with 0.1% 107.2 H₂O₂

As shown in Table III, with 20 parts of Perform® PC8229 (cationicfixative polymer) formulated with 18 parts of laccase NS51003(comparative example 3-2), the laccase activity was enhanced by 4% overlaccase alone (Comparative example 3-1). When an additional 12 parts ofthe fixative polymer (Example 2-3) was added to the composition another4% improvement in laccase activity was observed, resulting in an 8%improvement in total activity over the laccase alone. Ascorbic acid orsalicylic acid at 0.5% (Example 2-10, 2-11) in the assay furtherimproved the activity by about 7% with an overall improvement at 15%.Hydrogen peroxide did not help improve laccase activity (Example 3-1).

Copper ion such as that from copper sulfate was found to improve theactivity of laccase alone after the laccase enzyme was diluted. With0.05% to 0.1% of copper sulfate, the laccase activity improved more than30% while it was negatively affected when the dosage of copper sulfatewas higher than 0.5%. The effect of copper sulfate in the composition onlaccase activity is not significant.

Example 4—Performance of Enzyme Composition on Dry Strength of PaperMade from UBSK/TOCC Fiber Mix Via the Pilot Paper Machine Trial

Example 4, shows the improvement in dry strength properties of a papersheet made from unbleached softwood Kraft (UBSK)/TOCC fiber mix (25:75)by treating the lignocellulosic fiber with the dry strength compositionof the current method. The UBSK was pulped and refined to 475 ML CSFusing a circle beater and then blended with TOCC (CSF 300 ML) in theslurry chest at a temperature of about 50° C. to 60° C. The mixedfurnish was transferred to a machine chest of a pilot paper machine(located at the Hercules Research Center in Wilmington, Del.) and thentreated with the dry strength composition at 0.4% dosage based on thedry fiber for 15 minutes at 55° C. with agitation. The treated furnishor slurry was transferred to a small machine chest and used to producepaper sheets having a basis weight of 80 lb./3000 sq. ft. The MullenBurst and Ring Crush properties of the paper sheets were measured,normalized and expressed as versus the blank sheet made from UBSK/TOCCfiber mix at the 50/50 ratio with no dry strength additive (see TableIV).

TABLE IV Strength performance of fiber substitution with the enzymecomposition Fiber Enzyme Ring Mullen Examples composition compositionCrush % Burst % Comparative UBSK/TOCC None 100 100 Example 4-1 (50/50)Comparative UBSK/TOCC None 95.2 86.5 Example 4-1 (25/75) Example 4-1UBSK/TOCC Example 103.3 89.5 (25/75) 2-12

The UBSK/TOCC fiber mix (50:50) blank was a benchmark in this pilotpaper machine trial (Comparative example 4-1). Substituting 50% UBSKwith TOCC resulted in 5% lower in Ring Crush and 14% lower in MullenBurst (Comparative example 4-2). The dry strength composition of thecurrent method (Example 2-12) had 8% higher Ring Crush and 3% higherMullen Burst than the control without the treatment (Example 4-1). Thetreated furnish also achieved 50% UBSK reduction and was 3% higher inRing Crush.

Example 5—Effect of Mechanical Refining on Enzyme Compositions'Performance in Paper Dry Strength

Example 5, shows the improvement in dry strength properties of a papersheet made from AOCC by treating the fiber with two dry strengthcompositions of the current method either prior to or after mechanicalrefining. For the pre-refining experiment, the pulp slurry was incubatedwith the dry strength compositions for 1 hour at 60° C., and thenrefined to 300 CSF using a PFI mill. For post-refining treatment, thepulp slurry was first refined to 300 CSF using a PFI mill and then theresulting pulp slurry was treated with the dry strength compositions.The dosage difference in the two enzyme compositions was determinedbased on approximately equal costs of the two compositions. The treatedpulp furnish was used to make handsheets having a basis weight of 80lb./3000 sq. ft. The Mullen Burst and Ring Crush of the handsheets weremeasured and expressed as % versus the corresponding blanks for thepre-refining furnish and the post-refining furnish. Results aresummarized in Table V below.

TABLE V Strength performance of the enzyme composition on OCC before andafter refining Refining Enzyme after the Mullen Ring Examplescomposition treatment Dosage Burst % Crush % Example 5-1 Example 2-3 PFImill 0.25 105.4 117.2 Example 5-2 Example 2-1 PFI mill 0.3 106.3 110.8Example 5-3 Example 2-3 No 0.25 102.5 113.3 Example 5-4 Example 2-1 No0.3 103.1 111.2

The results show good improvement in Ring Crush (>10%) with the AOCCfurnish whether the dry strength composition was added to the furnishpre-refining or post refining of the furnish (Example 5-1 to 5-4). Theimprovement in Mullen Burst was less but pre-refining treatment appearedto give better Mullen burst strength properties at 5% to 6% over theblank (Example 5-1, 5-2).

Example 6—Performance of the Enzyme Compositions with Different Types ofOCC Furnishes

In this experiment, TOCC and COCC, an OCC recycled fiber from Asia wasused. This recycled fiber is of poor quality and a CSF<300 ML, whileAOCC is a much better quality OCC fiber with freeness in the range from400 to 600 ML CSF. The TOCC furnish also contains a lot of organicstickies and pitches. Handsheet experiments were done using two drystrength compositions made according to the current method wereevaluated on the poor quality TOCC and better quality AOCC for drystrength performance. AOCC furnish was refined to a pulp slurry of 300ML CSF using a PFI mill and was then treated with the dry strengthcompositions. The TOCC slurry was treated with the dry strengthcompositions without mechanical refining. All the treated pulp furnishwas used to make handsheets having a basis weight of 80 lb./3000 sq. ft.The Mullen Burst, Ring Crush and/or dry tensile of the handsheets weremeasured and expressed as % versus the blanks without enzyme treatment(see Table VI).

TABLE VI Performance of the enzyme compositions on TOCC and AOCC EnzymeDosage Mullen Dry Ring Exam- com- OCC wt. % Burst Tensile Crush plesposition furnish vs. fiber % % % Example Example TOCC 0 100 100 100 6-12-1 0.15 101.2 100.6 106.5 0.3 103.8 105.6 112.2 Example Example AOCC 0100 100 6-2 2-1 0.1 104.2 108 0.2 106.1 117.5 0.4 107.9 116.4 ExampleExample TOCC 0 100 100 100 6-3 2-3 0.15 107.5 113.5 103.0 0.3 109.2115.1 105.5

Table VI shows the results of a handsheet trial using two OCC fibershaving different paper dry strength properties and two dry strengthcompositions. These experiments demonstrated different performance ofthe two different enzyme compositions with TOCC. The enzyme compositionwith different weight percentages of laccase vs. lipase (i.e. 18% vs 12%(Example 2-3)) gave better performance in dry tensile and Mullen burstwith TOCC (15% improvement in Mullen Burst at 0.3% dosage (Example 6-3))while the dry strength composition with higher levels of laccase and theweight % of laccase vs. lipase at 24% to 6% (Example 2-1) had betterperformance in Ring Crush, giving 12% improvement at 0.3% dosage butless improvements in dry tensile and Mullen Burst (Example 6-1).

Each reference cited in the present application above, including books,patents, published applications, journal articles and otherpublications, is incorporated herein by reference in its entirety.

What is claimed is:
 1. A method of making a paper product with improveddry strength comprising: providing a pulp furnish or suspension having atemperature of from about 20° C. to about 70° C. and pH of from about4.0 to 9.0; treating the pulp furnish or suspension with a compositioncomprising from about 3 wt. % to about 40 wt. % dry weight of totalcomposition laccase and from about 1 wt. % to about 80 wt. % dry weightof total composition lipase, wherein the laccase has at least 12 LAMU oflaccase activity and the lipase has from about 0.1 to about 10 KLU oflipase activity per Kg of dry fiber, and wherein the composition isadded over a period of at least 0.1 hours; optionally refining thetreated pulp furnish or suspension using a mechanical refiner for woodfiber; optionally adding additional papermaking additives to the pulpfurnish; and drying and forming the pulp furnish into the desired paperproduct.
 2. The method of claim 1, wherein pulp furnish or suspension isrecycled OCC fiber.
 3. The method of claim 2, wherein the optionalpapermaking additive is selected from the group consisting of starch,starch derivatives, polyacrylamide derivatives, guar, poly(vinylamine),polyethyleneimine, urea formaldehyde resin, epichlorohydrin reactedpoly(aminoamide), starch aldehyde and glycoxylated polyacrylamide,flocculants, retention aids, drainage aids, debonders, softeners, sizingagents for paper products, and creping adhesives and enzymes.
 4. Themethod of claim 3, wherein the enzymes are selected from the groupconsisting of cellulases, hemicellulases, amylases, proteases, lipases,esterases, pectinases, lyases, pectate lyase, cellulase,oxidoreductases, glucose oxidases, and peroxidases.
 5. The methodaccording to claim 1, wherein the dry strength composition can be addedto the papermaking process either before, during or after mechanicalrefining in a papermaking process.